Preliminary and published results provide strong evidence that engineering short-lived Gut K-Cells to express the insulin gene is a potential therapy to treat type 1 diabetes mellitus (T1DM). Transgenic technologies are not amenable for human gene therapy, and a mechanism to stably introduce transgenes into small intestinal [epithelial] stem cells of adults is required. All published studies have demonstrated inefficient transduction of this stem cell population in vivo. Organoids containing functional intestinal epithelial stem cells can be isolated from rat small intestine and then grafted back onto denuded colonic muscle. The neomucosa exhibits a small intestinal, rather than colonic, phenotype. Grafts generated within the PI's laboratory contained Gut K-Cells, a cell normally present in the proximal small intestine and absent from colon. Thus, transplanted stem cells are hardwired to express genes from their original position along the duodenal to colonic axis. This suggests that organoids isolated from the proximal small intestine could be genetically modified before grafting back into the host and if so, could be engineered so Gut K-Cells derived from these stem cells could express the human insulin gene. Initial studies to test the feasibility of this strategy will determine whether the intestinal epithelial stem cells within the organoids can be genetically modified ex vivo by retroviral vectors (encoding beta-galactosidase) and then transplanted onto denuded colonic muscle. Grafts will also be assessed to determine whether beta-galactosidase expression is maintained for extended periods of time (9 months). Since retroviral vectors only infect proliferating cells, agents known to enhance crypt cell proliferation and/or inhibit apoptosis will be used in conjunction with viral infection in an attempt to increase the efficiency of stem cell transduction. Organoids can be grafted onto synthetic biodegradable polymers instead of colonic muscle and then anastomosed into the jejunum where the grafts remain intact for >9 months. If grafts containing genetically engineered Gut K-Cells were anastomosed back into the jejunum, they would be positioned to respond to normal metabolic and secretory signals. Thus, engineered Gut K-Cells would secrete insulin at the proper time to treat T1DM. Studies will be conducted to bring this alternative technique to the laboratory. Results from these pilot studies should provide a sound basis for devising a strategy to isolate, genetically modify and then transplant adult human intestinal epithelial stem cells as a potential treatment for T1DM.